Fig. 1
From: PRC2 specifies ectoderm lineages and maintains pluripotency in primed but not naïve ESCs
Deletion of polycomb repressive complex 2 in human embryonic stem cells. a Overview of the gene targeting strategy. gRNA was designed and validated for each polycomb repressive complex 2 (PRC2) component gene showing in box. To delete the critical domain for each factor, a homologous targeting vector containing puromycin or neomycin resistant cassette was constructed according to each gene. gRNA/Cas9 together with targeting vector were electroplated into H1 or H9 human embryonic stem cells (hESCs) and selected by the corresponding drug in defined condition. Positive clones were then isolated and expanded for further characterizations. b Targeting efficiencies of each gene. The functional domain that was deleted in each factor was shown. For SUZ12 and EED, gene targeting was performed in both H1 and H9 hESCs. c Morphology of H1 hESCs with targeted deletion of each gene. Scale bar, 200 μm. d qRT-PCR analysis on the expression level of each indicated gene in gene targeted hESCs. Wild-type H1 hESCs serve as control. Significance level was determined using unpaired two-tailed Student’s t tests. **, P < 0.01. The data represent mean ± SD from three biological repeats. e Total level of the indicated histone modification in gene targeted cells. The total histone modification level was analyzed by western-blot using the specific antibody on the whole-cell lysates from each indicated cell line. f qRT-PCR analysis on the expression level of the pluripotent genes, OCT4, SOX2, NANOG in gene targeted hESCs. Wild-type H1 hESCs serve as control. Significance level was determined using unpaired two-tailed Student’s t tests. **, P < 0.01. The data represent mean ± SD from three biological repeats. See also Supplementary Fig. 1
